Field of the Invention
The invention relates generally to the prediction of drug response and monitoring a disease state in a subject and more specifically to functional stratification of and signaling profiles of cancer cells upon modulation.
Background Information
Traditional pathological samples have been largely processed using methods that involve killing the cells using processing techniques that compromise the biological integrity of the sample. Such methods are generally performed in a laboratory well away from the point of care. These traditional methods do not permit the examination of live cells, including dynamic, live-cell related biomarkers, and do not allow for rapid sample processing or analytical result generation at or near the point of care. This lack of complete and rapidly obtained information can prevent doctors from identifying the proper treatment regimen or at the least slow the process which adversely affects the patient's quality of life.
For example, oncologists have a growing number of treatment options available to them, including different combinations of drugs that are characterized as standard of care, and a number of drugs that do not carry a label claim for a particular cancer, but for which there is evidence of efficacy in that cancer. The best likelihood of good treatment outcome requires that patients be assigned to optimal available cancer treatment, and that this assignment be made as quickly as possible following diagnosis.
While some cancers are beginning to be subclassified and treated using genomic markers, reliable genomic markers are not available for all cancers, which may be better characterized as exhibiting abnormal expression of one or (typically) many normal genes. Currently available biomarker tests to diagnose particular types of cancer and evaluate the likely effectiveness of different treatment strategies based on gene expression may have one or more disadvantages, for example: (1) the tests may be designed for testing blood and are not readily adapted for testing solid tumors; (2) sample preparation methods for solid tumor samples, may be unsuitable for handling live cells or performing subsequent measurements of marker expression; (3) small samples, e.g., obtained using fine needle biopsies, may not provide sufficient tissue for complete analysis; (4) the tests may require in vitro culturing of the cells, extended incubation periods, and/or significant delays between the time that the test cells are obtained from the patient and the time the cells are tested, resulting potential for wide variation and external influences on marker expression; (5) the tests may be unsuited for measuring expression of a multiplicity of genes, phosphoproteins or other markers in parallel, which may be critical for recognizing and characterizing the expression as abnormal; (6) the tests may be non-quantitative, relying principally on immunohistochemistry to determine the presence or absence of a protein as opposed to relative levels of expression of genes; (7) the reagents and cell handling conditions are not strictly controlled, leading to a high degree of variability from test to test and lab to lab; (8) the tests may be unsuited to analyzing nucleic acid levels, due to the instability of nucleic acid molecules and the practical difficulty of obtaining sufficiently fresh samples from the patients; and (9) the tests may involve fixing of the cells before any gene expression analysis can be performed, e.g., in the presence or absence of selected reagents.
Recently, several groups have published studies concerning the classification of various cancer types by microarray gene expression analysis (see, e.g. Golub et al., Science 286:531-537 (1999); Bhattacharjae et al., Proc. Nat. Acad. Sci. USA 98:13790-13795 (2001); Chen-Hsiang et al., Bioinformatics 17 (Suppl. 1): S316-S322 (2001); Ramaswamy et al., Proc. Natl. Acad. Sci. USA 98:1514915154 (2001)). Certain classifications of human breast cancers based on gene expression patterns have also been reported (Martin et al., Cancer Res. 60:2232-2238 (2000); West et al., Proc. Natl. Acad. Sci. USA 98:11462-11467 (2001); Sorlie et al., Proc. Natl. Acad. Sci. USA 98:1086910874 (2001); Yan et al., Cancer Res. 61:8375-8380 (2001)). However, these studies mostly focus on improving and refining the already established classification of various types of cancer, including breast cancer, and generally do not provide new insights into the relationships of the differentially expressed genes or functional cellular information. These studies do not link the findings to treatment strategies in order to improve the clinical outcome of cancer therapy, and they do not address the problem of improving and standardizing existing techniques of cell handling and analysis.
Although modern molecular biology and biochemistry have revealed more than 100 genes whose activities influence the behavior of tumor cells, state of their differentiation, and their sensitivity or resistance to certain therapeutic drugs, with a few exceptions, the status of these genes has been insufficient for the purpose of routinely making clinical decisions about drug treatments. One notable exception is the use of estrogen receptor (ER) protein expression in breast carcinomas to select patients to treatment with anti-estrogen drugs, such as tamoxifen. Another exceptional example is the use of ErbB2 (Her2) protein expression in breast carcinomas to select patients with the Her2 antagonist drug HERCEPTIN®. (Genentech, Inc., South San Francisco, Calif.). For most cancers, however, the pathologies in gene expression may be subtler and may involve patterns of expression of multiple genes or expression of genes in response to particular stimuli.
A tumor cell's response to a targeted therapeutic drug is dependent not only on the presence of the target, but also to the multitude of molecules, and their variants, within the signaling network. The term “ex vivo biomarker” defines a novel class of biomarkers—those which are evoked by live tumor cells after they have been removed from the patient. In the context of molecular biomarkers this refers to the process of removing viable cells from a patient through peripheral blood or bone marrow collection, during surgery, circulating tumor cells, or through a minimally-invasive biopsy such as a fine needle aspiration biopsy (FNA). The viable sample is then stimulated in vitro. In oncology applications these stimuli may be growth factors, such as epidermal growth factor, that are relevant to the signal transduction networks targeted by new therapeutic drugs. The biomarkers themselves can represent any dynamic biomolecule, but may be newly modified phosphoproteins or newly expressed mRNAs in the signaling network. Cellular events occurring rapidly (minutes) after ex vivo stimulation, such as protein phosphorylation events, may be considered “proximal” to the stimulus and may be most valuable in determining the dominant signal transduction pathways utilized by the tumor. Events occurring later following ex vivo stimulation (minutes to hours), such as new mRNA transcription, may be considered “distal” markers and may be more useful in assessing a composite view of the signal transduction events and their impact on cellular functions such as proliferation or apoptosis. Multiplexed panels of such phosphoproteins, or gene expression microarrays, may facilitate the generation of comprehensive functional profiles that are distinct from, and more informative than profiles generated from fixed tissues. In some cases the effect of a molecularly targeted agent (MTA) on the pathway could be monitored ex vivo by stimulating the sample in the presence of a modulator, such as a chemical pathway inhibitor or the MTA itself. Overall, ex vivo biomarkers offer the possibility of functional assays that interrogate entire signal transduction networks. Such assays offer several possible applications, including patient stratification based on functional information to inform clinical trial design or clinical management and novel pharmacodynamic assays for use in the development of targeted therapies. (Clark D P. Ex vivo biomarkers: functional tools to guide targeted drug development and therapy. Expert Rev Mol Diagn 2009; 9(8):787-94).
Thus, there remains a need to develop improved compositions and methods for diagnosing disease status and determining drug sensitivity of cancer cells based on functional stratification and/or signaling profiles.